Phenotypic consequences of LYS4 gene disruption in Candida albicans.

A BLAST search of the Candida Genome Database with the Saccharomyces cerevisiae LYS4 sequence known to encode homoaconitase (HA) revealed ORFs 19.3846 and 19.11327. Both alleles of the LYS4 gene were sequentially disrupted in Candida albicans BWP17 cells using PCR-based methodology. The null lys4Δ mutant exhibited lysine auxotrophy in minimal medium but was able to grow in the presence of l-Lys and α-aminoadipate, an intermediate of the α-aminoadipate pathway, at millimolar concentrations. The presence of d-Lys and pipecolic acid did not trigger lys4Δ growth. The C. albicans lys4Δ mutant cells demonstrated diminished germination ability. However, their virulence in vivo in a murine model of disseminated neonatal candidiasis appeared identical to that of the wild-type strain. Moreover, there was no statistically significant difference in fungal burden of infected tissues between the strains.

Association Between Antibiotic Duration and Recurrence of Urinary Tract Infection in the Neonatal Critical Care Unit.

OBJECTIVES: Urinary tract infection (UTI) is the most common bacterial infection in infants. Current practice guidelines suggest a treatment duration of 7 to 14 days. Suboptimal therapy may increase the risk for recurrent UTIs leading to renal scarring and possibly chronic kidney disease. The primary objective is to evaluate the duration of therapy for UTIs and its association with the incidence of recurrent UTIs in a neonatal intensive care unit (NICU). The secondary objectives are to identify the risk factors and the most common organisms for recurrent UTIs. METHODS: Patients were identified via the diagnosis codes for UTIs and were included if admitted to the NICU and if they received antibiotics prior to hospital discharge. Patients were divided into 2 groups: antibiotic treatment for 7 days or fewer and antibiotic treatment for greater than 7 days. RESULTS: Eighty-six infants were included in the study. Twenty-six patients received antibiotics for 7 days or fewer, and 60 for more than 7 days. In the study, the median birth weight was 977 g and the median gestational age was 27.6 weeks. There was no significant difference in the rate of recurrent UTIs between the 2 groups (p = 0.66). However, in the subgroup analysis, the incidence was higher for patients receiving antibiotic therapy for fewer than 7 days versus 7 days (p = 0.03). CONCLUSION: There was no difference in recurrence of UTI between treatment groups (≤7 days versus >7 days), and recurrence was seen in a higher percentage of patients with a urinary tract anomaly.

Prevalence and genetic analysis of phenotypically Vi- negative Salmonella typhi isolates in children from Kathmandu, Nepal.

The Vi capsular polysaccharide (ViPS) protects Salmonella enterica subspecies enterica serotype Typhi (S.Typhi) in vivo by multiple mechanisms. Recent microbiological reports from typhoid endemic countries suggest that acapsulate S.Typhi may occur in nature and contribute to clinical typhoid fever that is indistinguishable from disease caused by capsulate strains. The prevalence and genetic basis of ViPS-negative S.Typhi isolates in children from Kathmandu, Nepal, were tested in 68 isolates. Although 5.9% of isolates tested negative for capsular expression by slide agglutination tests, a novel multiplex PCR assay and individual PCR analyses demonstrated the presence of all 14 genes responsible for the synthesis, transportation and regulation of the ViPS. These data suggest that phenotypically acapsulate S.Typhi may not have a genetic basis for the same.

Appearance of peripheral blood plasma cells and memory B cells in a primary and secondary immune response in humans.

In humans, the kinetics of the appearance of memory B cells and plasma cells during primary immunization are not well defined. In this study, we assessed the primary B-cell response of rabies-antigen naive volunteers during a 3-dose course of rabies vaccine compared with the B-cell response to a booster dose of rabies vaccine given to previously immunized volunteers. After a single dose of vaccine, in the naive group plasma and memory B cells appeared later (peak at day 10) than in the primed group (peak at day 7) and were at lower frequency. The most rapid responses (day 4) were detected after a third immunization in the naive group. This is the first study to document the detailed kinetics of the plasma cell and memory B-cell responses to immunization in adult humans and to demonstrate differences in the responses that relate to the preexisting immune status of the persons.

High-throughput bacterial SNP typing identifies distinct clusters of Salmonella Typhi causing typhoid in Nepalese children.

BACKGROUND: Salmonella Typhi (S. Typhi) causes typhoid fever, which remains an important public health issue in many developing countries. Kathmandu, the capital of Nepal, is an area of high incidence and the pediatric population appears to be at high risk of exposure and infection. METHODS: We recently defined the population structure of S. Typhi, using new sequencing technologies to identify nearly 2,000 single nucleotide polymorphisms (SNPs) that can be used as unequivocal phylogenetic markers. Here we have used the GoldenGate (Illumina) platform to simultaneously type 1,500 of these SNPs in 62 S. Typhi isolates causing severe typhoid in children admitted to Patan Hospital in Kathmandu. RESULTS: Eight distinct S. Typhi haplotypes were identified during the 20-month study period, with 68% of isolates belonging to a subclone of the previously defined H58 S. Typhi. This subclone was closely associated with resistance to nalidixic acid, with all isolates from this group demonstrating a resistant phenotype and harbouring the same resistance-associated SNP in GyrA (Phe83). A secondary clone, comprising 19% of isolates, was observed only during the second half of the study. CONCLUSIONS: Our data demonstrate the utility of SNP typing for monitoring bacterial populations over a defined period in a single endemic setting. We provide evidence for genotype introduction and define a nalidixic acid resistant subclone of S. Typhi, which appears to be the dominant cause of severe pediatric typhoid in Kathmandu during the study period.

Biliary cirrhosis in a child with inherited interleukin-12 deficiency.

Interleukin-12 (IL-12) is a key cytokine in the defense against intracellular bacteria notably Mycobacteria and Salmonella species. We report a case of disseminated mycobacterial infection, following BCG vaccination, in a child who later developed tuberculosis. Functional tests and a novel diagnostic polymerase chain reaction (PCR) assay, revealed a loss-of-function deletion in the IL12 gene. Analysis of samples from the parents and siblings of the patient indicated an autosomal recessive inheritance pattern with varying degrees of phenotypic expression in identical genotypes. Interferon-gamma (IFN-gamma) therapy was associated with marked clinical improvement. Biliary cirrhosis, a hitherto unreported complication of IL-12 deficiency, developed later and required liver transplantation. A defect in the IL-12-IFN-gamma pathway should be suspected in patients presenting with multiple, repeated or persistent infection with intracellular bacteria. The diagnostic work-up and the immuno-genetic assay described here can aid in the quick and reliable diagnosis of IL-12 deficiency resulting from genetic defects and its subsequent management.

Comparison of the prevalence of tuberculosis infection in BCG vaccinated versus nonvaccinated school age children.

OBJECTIVE: To compare the prevalence of tuberculosis infection in BCG vaccinated versus non-vaccinated school age children in a tuberculosis endemic region. DESIGN: Cross-sectional, case control, school based survey. SETTING: Government lower primary school in Palakkad District, Kerala. METHODS: Tuberculosis infection was determined by tuberculin testing in 418 school children aged 5 to 9 years, utilizing a differential outcome variable definition for BCG vaccinated and unvaccinated children, in a tuberculosis endemic area with moderate vaccination coverage. Nutritional status was calculated using weight for age and weight for height criteria. RESULTS: Tuberculin positivity rate in unvaccinated children (24%) was significantly higher than in the vaccinated (9.7%) (P<0.001, RR: 2.9). Overall prevalence rate of tuberculosis infection was 15.5%. Boys had significantly higher vaccination rates than girls (P < 0.001). No association was found between tuberculin reaction size and age or nutritional status. CONCLUSIONS: BCG vaccination is associated with significant protection against the acquisition of Mycobacterium tuberculosis infection in childhood. This finding highlights the importance of universal implementation of BCG vaccination in children in tuberculosis endemic regions.

A National Survey of Neonatologists: Barriers and Prerequisites to Introduce Point-of-Care Ultrasound in Neonatal ICUs.

Point-of-care (POC) ultrasound refers to the use of portable imaging. Although POC ultrasound is widely available to the neonatologists in Australia and Europe, neonatologists in the United States report limited availability. Our objective was to seek the US neonatologists' perception of barriers and prerequisites in adopting POC ultrasound in neonatal intensive care units. An online survey link was sent via e-mail to 3000 neonatologists included in the database maintained by the American Academy of Pediatrics. Survey results (n = 574) were reported as percentage of total responses. Personal experience requiring an urgent sonography in managing cardiac tamponade or pleural effusion was reported by 78% respondents. However, emergent ultrasound (≤10 min) was not available in 80% of the neonatal intensive care units. We compared the responses based on years of clinical experience (>20 vs <20 years), with 272 (48%) neonatologist reporting more than 20 years of experience. Similarly, results from neonatal fellowship programs were compared with nonteaching/teaching hospitals, with 288 (50%) replies from neonatology fellowship programs. Compared with senior neonatologists, respondents with less than 20 years of clinical experience consider POC ultrasound enhances safety and accuracy of clinical procedures (87% vs 82%) and favor adopting POC ultrasound in clinical practice (92% vs 84%). There were no differences in opinion from neonatology fellowship programs compared with the nonteaching/teaching hospitals. Lack of training guidelines, inadequate support from local radiology department, and legal concerns were reported as the top 3 primary barriers in adopting POC ultrasound. If these barriers could be resolved, 89% respondents were inclined to adopt POC ultrasound in clinical practice.

The seroepidemiology of Haemophilus influenzae type b prior to introduction of an immunization programme in Kathmandu, Nepal.

Haemophilus influenzae type b (Hib) is now recognized as an important pathogen in Asia. To evaluate disease susceptibility, and as a marker of Hib transmission before routine immunization was introduced in Kathmandu, 71 participants aged 7 months-77 years were recruited and 15 cord blood samples were collected for analysis of anti-polyribosylribitol phosphate antibody levels by enzyme-linked immunosorbent assay. Only 20% of children under 5 years old had levels considered protective (>0.15 µg/ml), rising to 83% of 15-54 year-olds. Prior to introduction of Hib vaccine in Kathmandu, the majority of young children were susceptible to disease.

Chorea and developmental regression associated with human herpes virus-6 encephalitis.

We report a 14-month old child with multiple episodes of febrile status epilepticus, followed by chorea and developmental regression, caused by human herpes virus-6 encephalitis. Chorea has been described as a complication of relapsing herpes simplex virus I infection, but not as a manifestation of human herpes virus-6 infection. It is uncertain whether the chorea was an autoimmune phenomenon or a direct effect of the virus. The child was treated with levetiracetam, intravenous immunoglobulin, and foscarnet. The seizures and chorea resolved with treatment, but developmental regression, with loss of language skills, persisted 6 months after the illness. This child illustrates a new clinical presentation of human herpes virus-6 encephalitis, adds to the spectrum of disorders caused by this virus, and strengthens the case for routine identification of specific viral agents in all cases of childhood viral infections with central nervous system symptoms to determine optimal treatment and prognosis.

Kinetics of the natural, humoral immune response to Salmonella enterica serovar Typhi in Kathmandu, Nepal.

Typhoid fever is a major public health problem in developing countries, conservatively estimated to occur in 17 million cases and be responsible for 200,000 deaths annually. We investigated the acquisition of natural immunity to Salmonella enterica serovar Typhi in a region where typhoid is endemic by testing sera from an age-stratified sample of 210 healthy participants in Kathmandu, Nepal, for bactericidal activity toward S. Typhi and for anti-Vi capsular polysaccharide antibodies. Bactericidal titers in children were significantly lower than those in newborns and adults (P < 0.0001). Anti-S. Typhi bactericidal geometric mean titers were age dependent, increasing 10-fold during childhood. Anti-Vi polysaccharide antibody geometric mean concentrations were also lower in children than in adults. Data presented here indicate the possibility of a relationship between low levels of bactericidal activity toward S. Typhi in serum and susceptibility to disease, as observed for other polysaccharide-encapsulated bacteria. Bactericidal antibody may be a marker of protective immunity against S. Typhi.